Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm
Summary
Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.
Abstract
Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.
Introduction
Robotics have been established as sophisticated and powerful tools for achieving significant breakthroughs in various fields beyond industry and fabrication engineering, such as biochemistry, molecular biology, and clinical research, and notably HTS1,2,3. Toxoplasma gondii is a major parasite and a single-cell parasitic eukaryote4 that causes serious health issues in humans5 and many homeothermic animals4, resulting in infections leading to Toxoplasmosis, a particularly severe condition in AIDS patients6, organ transplant recipients7, and pregnant women8. Neospora caninum belonging to Phylum Apicomplexa9 mainly infects dogs and cows6, which results in Encephalomyelitis and Myositis-Polyradiculitis in dogs10,11 and abortion in cows12,13. Further, Neospora caninum exhibits morphological and phylogenetical close similarities of Toxoplasma gondii9,14. Additionally, they have a nucleoside triphosphate hydrolase (NTPase; EC3.6.1.15)14. The enzymes are quite different from conventional ecto-ATPase14. These parasites generate a considerable amount of NTPase proteins, 2%-8% of the total protein and play an important role as dormant enzymes in their tachyzoite stage15. It should be noted that in dense secretory granules, these are condensed16 and secreted into the parasitophorous vacuole16. As a biochemical enzymatic character, NTPase is activated by dithiothreitol17. It is predicted that the inducers such as the dithiol compound, an unidentified enzyme such as dithiol-disulfide oxidoreductase, and another exhibit the same nature. They have not yet been identified in parasites. However, the enzyme does play an important role in releasing tachyzoite from infected host cells17.
Toxoplasma gondii has two NTPase isoforms18: type I enzyme TgNTPase-I, and type II enzyme TgNTPase-II18. The former preferentially utilizes triphosphate nucleosides as a substrate18. The latter hydrolyzes both triphosphate and diphosphate nucleosides18. The homology is 97% in amino acid levels18. Neospora caninum also has an orthologue of TgNTPase-I named NcNTPase19. The homology is 73% in amino acid levels19. Prof. Asai and Prof. Harada generated recombinants of both the NTPase using E. coli. and changed the constitutively active mutants of these as previously reported20. They kindly gifted the two active mutants. Both enzymes can convert ATP to ADP in vitro20. Very recently, we measured the activity of NTPase using ADP content hydrolyzed by the enzymes. Finally, we succeeded in establishing the high-standard assay through the process of determining ADP content with a combination of fluorescence and enzymatic reaction as previously reported21,22. We also did high-throughput screening (HTS)22.
This study introduces detailed procedures of a novel high-accuracy and dynamic-range assay21 and a detailed explanation on how to prepare reagents to measure the enzyme activity and develop fluorescent intensity using a robot arm for HTS.
Protocol
Representative Results
Discussion
We succeeded in establishing a novel high-dynamic range and -accuracy assay with a combination of a classical enzyme assay and a fluorescent assay for ADP, which is the end product through ATPase, including Tg and Nc ATPase22. In order to carry out HTS, it is important that the assay has better values of S/B, S/N, and Z’ factor than a classical enzyme assay15,22. Additionally, omitting the step of stopping the enzyme reaction with an acid …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was partly supported by the Platform for Drug Discovery, Informatics and Structural Life Science, a Grant-in-Aid for Scientific Research (C) from Japan Society for Promotion of Science (JSPS-21K06566). The authors sincerely thank Asai (Keio University School of medicine) and Harada (Kyoto Institute of Technology) and Stephen Stratton for gifting recombinant two NTPase active mutants and his contribution in the preparation of this manuscript, respectively.
Materials
| 12 stage-workstation EDR-384 SX | Biotec Co., Ltd. | EDR-384SX | Robot arm Pippeting system |
| 384 well tips | Biotech Co., Ltd. | Custom made | |
| ADP-hexokinase | Asahi Kasei Pharma Co., Ltd. | T-92 | |
| ATP | Oriental Yeast, Co, Ltd. | 45140000 | |
| BSA | Wako Pure Chemical Industries, Ltd. | 011-15144 | |
| Diaphorase-I | Unitika Ltd. | Di-1 | |
| DMSO | Nacalai Tesch, Inc. | 13406-55 | |
| G6P dehydrogenase | Oriental Yeast, Co, Ltd. | 306-50143 | |
| Glucose | Wako Pure Chemical Industries, Ltd. | 049-31165 | |
| Greiner 384 well micro-plate non-binding shallow well Black | #784900 | ||
| HEPES | Wako Pure Chemical Industries, Ltd. | 342-01375 | |
| Mg(CH3COO)2 | Wako Pure Chemical Industries, Ltd. | 130-00095 | |
| NADP | Oriental Yeast, Co, Ltd. | 44332000 | |
| N-ethylmaleimide | Wako Pure Chemical Industries, Ltd. | 056-02062 | |
| PHERAstar FS | BMG LABTECH JAPAN L.t.d. | PHERAstar FS | Multimode microplate reader |
| Resazurin | Wako Pure Chemical Industries, Ltd. | 191-07581 | |
| Seahorse Labware 384 Well Low profile reservoirs | S30022 25/CS | ||
| TrisHCl | Wako Pure Chemical Industries, Ltd. | W01COBQE-4120 | |
| Triton X-100 | Nacalai Tesch, Inc. | 35501-02 |
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